Chlamydia trachomatis serovars D-K are the most common cause, in the USA and worldwide, of bacterially acquired sexually transmitted diseases and their sequelae, including prostatitis, epididymitis, pelvic inflammatory disease, ectopic pregnancy and sterility. Chlamydial diseases are insidious and they constitute significant primary, secondary and tertiary health concerns in which women bear a special burden because of increased risk of adverse reproductive consequences. The goal of our laboratory for 25 years has been to try to understand the basic biology of Chlamydial growth in its host epithelial cell in order to understand the infectious process and to permit dissection of the cellular/molecular consequences of persistent infection, since the majority of Chlamydial tubal disease appears to result from subclinical, persistent infection. This proposal is a continuation of on-going efforts to understand the crucial attachment steps, which are modulated by female hormones in vitro. In Specific Aim 1, we shall determine whether or not estrogen receptors (ER) are involved in serovar E attachment to human endometrial epithelial cells or, alternatively, if the estrogen-induced physiological effects on the host cell result in display of other receptors. Specific labeled-EB binding assays to ER+ cell lines will be used in addition to BiaCore, FACS, and microscopy analyses and coupled with siRNA technology to knockdown surface ER amounts to see if reduced EB binding does occur. In Specific Aim 2, we shall monitor, by microscopy and gradient vesicle isolation, mechanisms of serovar E outer membrane bleb antigen escape through the inclusion membrane as well as from inclusion membrane everted vesicles and determine trafficking, including endoplasmic reticulum membrane fusion potential for MHC Class I loading. Finally, for Specific Aim 3, we have isolated Chlamydial projections (small scale) and, after bulk collection and generation of antibodies, plan to characterize these surface structures; the obvious question is whether or not the projections are the Chlamydial Type III secretion injectisome-equivalent.